Peroxidase activity towards 10-acetyl-3,7-dihydroxyphenoxazine

ABSTRACT

The present invention provides engineered peroxidase enzymes, polypeptides having peroxidase activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing peroxidase enzymes are also provided. The present invention further provides compositions comprising the peroxidase enzymes and methods of using the engineered peroxidase enzymes. The present invention finds particular use in the production of pharmaceutical and other compounds.

The present application claims priority to U.S. Prov. Pat. Appln. Ser.No. 62/899,190, filed Sep. 12, 2019, which is incorporated by referencein its entirety, for all purposes.

FIELD OF THE INVENTION

The present invention provides engineered peroxidase enzymes,polypeptides having peroxidase activity, and polynucleotides encodingthese enzymes, as well as vectors and host cells comprising thesepolynucleotides and polypeptides. Methods for producing peroxidaseenzymes are also provided. The present invention further providescompositions comprising the peroxidase enzymes and methods of using theengineered peroxidase enzymes. The present invention finds particularuse in the production of pharmaceutical and other compounds.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-190USP1_ST25.txt”, a creation date of Sep. 11, 2019and a size of 852 kilo kilobytes. The Sequence Listing filed via EFS-Webis part of the specification and incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

Peroxidases, notably horse radish peroxidase (HRP), have been widelyused in immunohistochemistry applications and oxidation reactions. Horseradish peroxidase (EC 1.11.1.7) is an iron-dependent enzyme thatoxidizes organic substrates using hydrogen peroxide and is also known topromote oxidase catalytic activity by oxidizing an inactive redox stateof the active site during normal oxidase catalytic cycling. HRP is a 44kDa protein, where the polypeptide is 33 kDa, and glycosylation accountsfor the remaining mass. One drawback to using HRP for industrialapplications is the difficulty of efficiently expressing theheavily-glycosylated protein in bacterial systems. Thus, there is a needfor alternatives to HRP that are easily expressed and enable highyielding oxidase reactions. Alternative peroxidase enzymes may also haveutility in other oxidation chemistries.

SUMMARY OF THE INVENTION

The present invention provides engineered DyP-type peroxidase enzymes,polypeptides capable of oxidation of 10-acetyl-3,7-dihydroxyphenoxazine(Amplex Red) using hydrogen peroxide as the electron donor, andpolynucleotides encoding these enzymes, as well as vectors and hostcells comprising these polynucleotides and polypeptides. Methods forproducing peroxidase enzymes are also provided. The present inventionfurther provides compositions comprising the peroxidase enzymes andmethods of using the engineered peroxidase enzymes. The presentinvention finds particular use in the production of pharmaceutical andother compounds.

DyP-type peroxidase (DyP) (EC number 1.11.1.19) is a family of dyede-colorizing peroxidase enzymes. The enzyme is a haem containingprotein that catalyzes the oxidation of a number of substrates usinghydrogen peroxide as the electron donor. The engineered DyP peroxidaseused in this process has a sequence derived from the sequence of thewild-type peroxidase Alr1585 from cyanobacteria Anabaena (Nostoc) sp.(strain PCC 7120).

The present invention provides engineered peroxidases comprisingpolypeptide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 4 and/or 6, or a functional fragment thereof. In someembodiments, the engineered peroxidase comprises at least onesubstitution or substitution set in the polypeptide sequence, andwherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 4 and/or 6. In some additionalembodiments of the engineered peroxidase, the polypeptide sequences haveat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4. In someembodiments, the engineered peroxidase comprises at least onesubstitution or substitution set at one or more positions in thepolypeptide sequence selected from 25/29/43, 25/29/65/82/92, 26/29,80/128/306, 90, 127/168, 127/301, 128/307, 143, 160, 160/188, 160/307,164, 168, 171/260, 185, 185/307, 189/190/226, 191, 191/260, 191/469,202, 206, 208, 221, 222, 224, 224/260, 226, 228, 228/260,241/270/283/306, 260/307, 290, 301, 306, 307, 311, 332, 367, and 393,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 4. In some embodiments, theengineered peroxidase comprises at least one substitution orsubstitution set selected from 25A/29A/43Y, 25Q/29A/65G/82K/92G,26E/29K, 80K/128K/306F, 90Q, 90Y, 127K/168S, 127K/301I, 128K/307N,128K/307Q, 143H, 143R, 143S, 143V, 160H/188V, 160M, 160R, 160V/307C,164D, 164K, 168H, 168K, 168M, 168S, 168W, 171V/260R, 185E/307H, 185Q,185T, 189W/190V/226P, 191A, 191D, 191G, 191S, 191S/260R, 191S/469V,202W, 206L, 208N, 221D, 221H, 221L, 221M, 221P, 221R, 221S, 221T, 221Y,222E, 222G, 222S, 222V, 224L, 224P, 224R, 224R/260R, 224Y, 226I, 226V,228G, 228M/260R, 241K/270N/283T/306L, 260R/307T, 290L, 301S, 301V, 301W,306A, 306K, 306V, 307C, 307D, 307E, 307G, 307L, 307M, 307R, 307S, 311I,311V, 332A, 367G, and 393D, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 4. Insome additional embodiments, the engineered peroxidase comprises atleast one substitution or substitution set selected from R25A/N29A/H43Y,R25Q/N29A/S65G/F82K/D92G, N26E/N29K, E80K/E128K/T306F, S90Q, S90Y,E127K/N168S, E127K/L301I, E128K/Y307N, E128K/Y307Q, L143H, L143R, L143S,L143V, I160H/I188V, I160M, I160R, I160V/Y307C, L164D, L164K, N168H,N168K, N168M, N168S, N168W, T171V/L260R, D185E/Y307H, D185Q, D185T,L189W/R190V/F226P, N191A, N191D, N191G, N191S, N191S/L260R, N191S/A469V,F202W, V206L, Q208N, V221D, V221H, V221L, V221M, V221P, V221R, V221S,V221T, V221Y, N222E, N222G, N222S, N222V, C224L, C224P, C224R,C224R/L260R, C224Y, F226I, F226V, K228G, K228M/L260R,E241K/D270N/N283T/T306L, L260R/Y307T, V290L, L301S, L301V, L301W, T306A,T306K, T306V, Y307C, Y307D, Y307E, Y307G, Y307L, Y307M, Y307R, Y307S,P311I, P311V, T332A, A367G, and N393D, wherein the amino acid positionsof the polypeptide sequence are numbered with reference to SEQ ID NO: 4.

In some additional embodiments, the engineered peroxidase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 6, and wherein the engineered peroxidase comprises at least onesubstitution or substitution set at one or more positions in thepolypeptide sequence selected from 25/29/43, 25/29/65/82/92, 80/128/306,90, 127/301, 128/307, 143, 160, 160/188, 160/307, 164, 168, 171/260,185, 185/307, 191, 191/260, 191/469, 202, 206, 208, 221, 222, 224,224/260, 228, 228/260, 241/270/283/306, 260/307, 301, 306, 307, 311,332, 367, and 393, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 6. In someembodiments, the engineered peroxidase comprises at least onesubstitution or substitution set selected from 25A/29A/43Y,25Q/29A/65G/82K/92G, 80K/128K/306F, 90Y, 127K/301I, 128K/307N,128K/307Q, 143H, 143R, 160H/188V, 160M, 160V/307C, 164D, 168W,171V/260R, 185E/307H, 185Q, 185T, 191D, 191G, 191S, 191S/260R,191S/469V, 202W, 206L, 208N, 221H, 221L, 221M, 221P, 221S, 221T, 221Y,222E, 222S, 224L, 224P, 224R, 224R/260R, 224Y, 228G, 228M/260R,241K/270N/283T/306L, 260R/307T, 301S, 301V, 306A, 306V, 307C, 307D,307E, 307G, 307L, 307M, 307R, 307S, 311V, 332A, 367G, and 393D, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 6. In some embodiments, the engineeredperoxidase comprises at least one substitution or substitution setselected from R25A/N29A/H43Y, R25Q/N29A/S65G/F82K/D92G,E80K/E128K/T306F, S90Y, E127K/L301I, E128K/Y307N, E128K/Y307Q, L143H,L143R, I160H/I188V, I160M, I160V/Y307C, L164D, N168W, T171V/L260R,D185E/Y307H, D185Q, D185T, N191D, N191G, N191S, N191S/L260R,N191S/A469V, F202W, V206L, Q208N, V221H, V221L, V221M, V221P, V221S,V221T, V221Y, N222E, N222S, C224L, C224P, C224R, C224R/L260R, C224Y,K228G, K228M/L260R, E241K/D270N/N283T/T306L, L260R/Y307T, L301S, L301V,T306A, T306V, Y307C, Y307D, Y307E, Y307G, Y307L, Y307M, Y307R, Y307S,P311V, T332A, A367G, and N393D, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 6.

In yet some additional embodiments, the engineered peroxidase comprisesa polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to thesequence of at least one engineered peroxidase variant set forth inTable 5.1 and/or 6.1 In still some further embodiments, the engineeredperoxidase comprises a polypeptide sequence that is at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to the sequence of at least one engineered peroxidase variantset forth in SEQ ID NOS: 4 and/or 6. In some embodiments, the engineeredperoxidase is a variant engineered peroxidase set forth in SEQ ID NOS: 4and/or 6. In some further embodiments, the engineered peroxidasecomprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the sequence of at least one engineered peroxidase variant set forthin the even numbered sequences of SEQ ID NOS: 6-290. In still someadditional embodiments, the engineered peroxidase comprises apolypeptide sequence set forth in the even numbered sequences of SEQ IDNOS: 6-290. In some further embodiments, the engineered peroxidasecomprises at least one improved property compared to wild-type Anabaena(Nostoc) sp. (strain PCC 7120) peroxidase. In some embodiments, theimproved property comprises improved activity on a substrate. In someadditional embodiments, the substrate comprises10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). In yet some additionalembodiments, the engineered peroxidase is purified. The presentinvention also provides compositions comprising at least one engineeredperoxidase provided herein.

The present invention also provides polynucleotide sequences encodingthe engineered peroxidases provided herein. In some embodiments, thepolynucleotide sequences encode more than one of the engineeredperoxidases provided herein. The present invention also providespolynucleotide sequences encoding at least one engineered peroxidase,wherein the polynucleotide sequence comprises at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NOS: 3 and/or 5, wherein the polynucleotidesequence of the engineered peroxidase comprises at least onesubstitution at one or more positions. In some additional embodiments,the polynucleotide sequence encoding at least one engineered peroxidasecomprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOS: 3and/or 5, or a functional fragment thereof. In some further embodiments,the polynucleotide sequence is operably linked to a control sequence. Insome additional embodiments, the polynucleotide sequence is codonoptimized. In yet some further embodiments, the polynucleotide comprisesan odd-numbered sequence of SEQ ID NOS: 5-289. The present inventionalso provides expression vectors comprising at least one polynucleotidesequence encoding at least one peroxidase provided herein. The presentinvention also provides host cells comprising at least one expressionvector provided herein. The present invention also provides host cellscomprising at least one polynucleotide sequence encoding at least oneperoxidase provided herein.

The present invention also provides methods of producing an engineeredperoxidase in a host cell, comprising culturing a host cell undersuitable conditions, such that at least one engineered peroxidaseprovided herein is produced. In some embodiments, the methods furthercomprise recovering at least one engineered peroxidase from the cultureand/or host cell. In some additional embodiments, the methods furthercomprise the step of purifying the at least one engineered peroxidaseprovided herein.

DESCRIPTION OF THE INVENTION

The present invention provides engineered peroxidase enzymes,polypeptides capable of oxidation of 10-acetyl-3,7-dihydroxyphenoxazine(Amplex Red) using hydrogen peroxide as the electron donor, andpolynucleotides encoding these enzymes, as well as vectors and hostcells comprising these polynucleotides and polypeptides. Methods forproducing peroxidase enzymes are also provided. The present inventionfurther provides compositions comprising the peroxidase enzymes andmethods of using the engineered peroxidase enzymes. The presentinvention finds particular use in the production of pharmaceutical andother compounds.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Generally,the nomenclature used herein and the laboratory procedures of cellculture, molecular genetics, microbiology, organic chemistry, analyticalchemistry and nucleic acid chemistry described below are thosewell-known and commonly employed in the art. Such techniques arewell-known and described in numerous texts and reference works wellknown to those of skill in the art. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses. All patents, patent applications, articles and publicationsmentioned herein, both supra and infra, are hereby expresslyincorporated herein by reference.

Although any suitable methods and materials similar or equivalent tothose described herein find use in the practice of the presentinvention, some methods and materials are described herein. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context they are used by those of skill in the art.Accordingly, the terms defined immediately below are more fullydescribed by reference to the invention as a whole.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the present invention. The section headingsused herein are for organizational purposes only and not to be construedas limiting the subject matter described. Numeric ranges are inclusiveof the numbers defining the range. Thus, every numerical range disclosedherein is intended to encompass every narrower numerical range thatfalls within such broader numerical range, as if such narrower numericalranges were all expressly written herein. It is also intended that everymaximum (or minimum) numerical limitation disclosed herein includesevery lower (or higher) numerical limitation, as if such lower (orhigher) numerical limitations were expressly written herein.

Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows: alanine (Ala or A), arginine (Arg orR), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C),glutamate (Glu or E), glutamine (Gln or Q), histidine (His or H),isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine(Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser orS), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y),and valine (Val or V).

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (Cα). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleosides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

Definitions

In reference to the present invention, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a polypeptide” includes more than onepolypeptide.

Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.Thus, as used herein, the term “comprising” and its cognates are used intheir inclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

As used herein, the term “about” means an acceptable error for aparticular value. In some instances, “about” means within 0.05%, 0.5%,1.0%, or 2.0%, of a given value range. In some instances, “about” meanswithin 1, 2, 3, or 4 standard deviations of a given value.

As used herein, “EC” number refers to the Enzyme Nomenclature of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (NC-IUBMB). The IUBMB biochemical classification is anumerical classification system for enzymes based on the chemicalreactions they catalyze.

As used herein, “ATCC” refers to the American Type Culture Collectionwhose biorepository collection includes genes and strains.

As used herein, “NCBI” refers to National Center for BiologicalInformation and the sequence databases provided therein.

As used herein, “horseradish peroxidase” (HRP, EC 1.11.1.7) enzyme is aniron-dependent enzyme that oxidizes a variety of organic substrates.While HRP is specifically referred to in the description includedherein, it is not meant to be an exclusive reference, as there are otherisoforms of this enzyme class and chemical reagents that have similarfunction.

As used herein, “improved peroxidase,” “engineered peroxidase,”“engineered peroxidase enzymes,” “peroxidase variants,” “DyP enzymes,”and the like refer to engineered DyP-type peroxidases. DyP-typeperoxidase (DyP) (EC number 1.11.1.19) is a family of dye de-colorizingperoxidase enzymes. The enzyme is a haem containing protein thatcatalyzes the oxidation of a number of substrates using hydrogenperoxide as the electron donor. The engineered DyP used in this processhas a sequence derived from the sequence of the wild-type peroxidaseAlr1585 from cyanobacteria Anabaena (Nostoc) sp. (strain PCC 7120).

“Amino acids” are referred to herein by either their commonly knownthree-letter symbols or by the one-letter symbols recommended byIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single letter codes.

As used herein, “hydrophilic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity of lessthan zero according to the normalized consensus hydrophobicity scale ofEisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142 [1984]).Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser(S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K)and L-Arg (R).

As used herein, “acidic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pKa value of lessthan about 6 when the amino acid is included in a peptide orpolypeptide. Acidic amino acids typically have negatively charged sidechains at physiological pH due to loss of a hydrogen ion. Geneticallyencoded acidic amino acids include L-Glu (E) and L-Asp (D).

As used herein, “basic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pKa value ofgreater than about 6 when the amino acid is included in a peptide orpolypeptide. Basic amino acids typically have positively charged sidechains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

As used herein, “polar amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain that is uncharged atphysiological pH, but which has at least one bond in which the pair ofelectrons shared in common by two atoms is held more closely by one ofthe atoms. Genetically encoded polar amino acids include L-Asn (N),L-Gln (Q), L-Ser (S) and L-Thr (T).

As used herein, “hydrophobic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity ofgreater than zero according to the normalized consensus hydrophobicityscale of Eisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142[1984]). Genetically encoded hydrophobic amino acids include L-Pro (P),L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala(A) and L-Tyr (Y).

As used herein, “aromatic amino acid or residue” refers to a hydrophilicor hydrophobic amino acid or residue having a side chain that includesat least one aromatic or heteroaromatic ring. Genetically encodedaromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W).Although owing to the pKa of its heteroaromatic nitrogen atom L-His (H)it is sometimes classified as a basic residue, or as an aromatic residueas its side chain includes a heteroaromatic ring, herein histidine isclassified as a hydrophilic residue or as a “constrained residue” (seebelow).

As used herein, “constrained amino acid or residue” refers to an aminoacid or residue that has a constrained geometry. Herein, constrainedresidues include L-Pro (P) and L-His (H). Histidine has a constrainedgeometry because it has a relatively small imidazole ring. Proline has aconstrained geometry because it also has a five membered ring.

As used herein, “non-polar amino acid or residue” refers to ahydrophobic amino acid or residue having a side chain that is unchargedat physiological pH and which has bonds in which the pair of electronsshared in common by two atoms is generally held equally by each of thetwo atoms (i.e., the side chain is not polar). Genetically encodednon-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile(I), L-Met (M) and L-Ala (A).

As used herein, “aliphatic amino acid or residue” refers to ahydrophobic amino acid or residue having an aliphatic hydrocarbon sidechain. Genetically encoded aliphatic amino acids include L-Ala (A),L-Val (V), L-Leu (L) and L-Ile (I). It is noted that cysteine (or“L-Cys” or “[C]”) is unusual in that it can form disulfide bridges withother L-Cys (C) amino acids or other sulfanyl- or sulfhydryl-containingamino acids. The “cysteine-like residues” include cysteine and otheramino acids that contain sulfhydryl moieties that are available forformation of disulfide bridges. The ability of L-Cys (C) (and otheramino acids with —SH containing side chains) to exist in a peptide ineither the reduced free —SH or oxidized disulfide-bridged form affectswhether L-Cys (C) contributes net hydrophobic or hydrophilic characterto a peptide. While L-Cys (C) exhibits a hydrophobicity of 0.29according to the normalized consensus scale of Eisenberg (Eisenberg etal., 1984, supra), it is to be understood that for purposes of thepresent disclosure, L-Cys (C) is categorized into its own unique group.

As used herein, “small amino acid or residue” refers to an amino acid orresidue having a side chain that is composed of a total three or fewercarbon and/or heteroatoms (excluding the α-carbon and hydrogens). Thesmall amino acids or residues may be further categorized as aliphatic,non-polar, polar or acidic small amino acids or residues, in accordancewith the above definitions. Genetically-encoded small amino acidsinclude L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T)and L-Asp (D).

As used herein, “hydroxyl-containing amino acid or residue” refers to anamino acid containing a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

As used herein, “polynucleotide” and “nucleic acid′ refer to two or morenucleotides that are covalently linked together. The polynucleotide maybe wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of2′ deoxyribonucleotides (i.e., DNA), or comprised of mixtures of ribo-and 2′ deoxyribonucleotides. While the nucleosides will typically belinked together via standard phosphodiester linkages, thepolynucleotides may include one or more non-standard linkages. Thepolynucleotide may be single-stranded or double-stranded, or may includeboth single-stranded regions and double-stranded regions. Moreover,while a polynucleotide will typically be composed of the naturallyoccurring encoding nucleobases (i.e., adenine, guanine, uracil, thymineand cytosine), it may include one or more modified and/or syntheticnucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.In some embodiments, such modified or synthetic nucleobases arenucleobases encoding amino acid sequences.

As used herein, “coding sequence” refers to that portion of a nucleicacid (e.g., a gene) that encodes an amino acid sequence of a protein.

As used herein, the terms “biocatalysis,” “biocatalytic,”“biotransformation,” and “biosynthesis” refer to the use of enzymes toperform chemical reactions on organic compounds.

As used herein, “wild-type” and “naturally-occurring” refer to the formfound in nature. For example, a wild-type polypeptide or polynucleotidesequence is a sequence present in an organism that can be isolated froma source in nature and which has not been intentionally modified byhuman manipulation.

As used herein, “recombinant,” “engineered,” ‘variant,” and“non-naturally occurring” when used with reference to a cell, nucleicacid, or polypeptide, refers to a material, or a material correspondingto the natural or native form of the material, that has been modified ina manner that would not otherwise exist in nature. In some embodiments,the cell, nucleic acid or polypeptide is identical a naturally occurringcell, nucleic acid or polypeptide, but is produced or derived fromsynthetic materials and/or by manipulation using recombinant techniques.Non-limiting examples include, among others, recombinant cellsexpressing genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise expressed ata different level.

The term “percent (%) sequence identity” is used herein to refer tocomparisons among polynucleotides or polypeptides, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage may be calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Alternatively, thepercentage may be calculated by determining the number of positions atwhich either the identical nucleic acid base or amino acid residueoccurs in both sequences or a nucleic acid base or amino acid residue isaligned with a gap to yield the number of matched positions, dividingthe number of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Those of skill in the art appreciatethat there are many established algorithms available to align twosequences. Optimal alignment of sequences for comparison can beconducted by any suitable method, including, but not limited to thelocal homology algorithm of Smith and Waterman (Smith and Waterman, Adv.Appl. Math., 2:482 [1981]), by the homology alignment algorithm ofNeedleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443[1970]), by the search for similarity method of Pearson and Lipman(Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]), bycomputerized implementations of these algorithms (e.g., GAP, BESTFIT,FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visualinspection, as known in the art. Examples of algorithms that aresuitable for determining percent sequence identity and sequencesimilarity include, but are not limited to the BLAST and BLAST 2.0algorithms, which are described by Altschul et al. (See Altschul et al.,J. Mol. Biol., 215: 403-410 [1990]; and Altschul et al., Nucl. AcidsRes., 3389-3402 [1977], respectively). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length Win the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (See, Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix(See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915[1989]). Exemplary determination of sequence alignment and % sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

As used herein, “reference sequence” refers to a defined sequence usedas a basis for a sequence and/or activity comparison. A referencesequence may be a subset of a larger sequence, for example, a segment ofa full-length gene or polypeptide sequence. Generally, a referencesequence is at least 20 nucleotide or amino acid residues in length, atleast 25 residues in length, at least 50 residues in length, at least100 residues in length or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptides aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence.

As used herein, “comparison window” refers to a conceptual segment of atleast about 20 contiguous nucleotide positions or amino acid residueswherein a sequence may be compared to a reference sequence of at least20 contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

As used herein, “corresponding to,” “reference to,” and “relative to”when used in the context of the numbering of a given amino acid orpolynucleotide sequence refer to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. In other words, theresidue number or residue position of a given polymer is designated withrespect to the reference sequence rather than by the actual numericalposition of the residue within the given amino acid or polynucleotidesequence. For example, a given amino acid sequence, such as that of anengineered peroxidase, can be aligned to a reference sequence byintroducing gaps to optimize residue matches between the two sequences.In these cases, although the gaps are present, the numbering of theresidue in the given amino acid or polynucleotide sequence is made withrespect to the reference sequence to which it has been aligned.

As used herein, “substantial identity” refers to a polynucleotide orpolypeptide sequence that has at least 80 percent sequence identity, atleast 85 percent identity, at least between 89 to 95 percent sequenceidentity, or more usually, at least 99 percent sequence identity ascompared to a reference sequence over a comparison window of at least 20residue positions, frequently over a window of at least 30-50 residues,wherein the percentage of sequence identity is calculated by comparingthe reference sequence to a sequence that includes deletions oradditions which total 20 percent or less of the reference sequence overthe window of comparison. In some specific embodiments applied topolypeptides, the term “substantial identity” means that two polypeptidesequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights, share at least 80 percent sequenceidentity, preferably at least 89 percent sequence identity, at least 95percent sequence identity or more (e.g., 99 percent sequence identity).In some embodiments, residue positions that are not identical insequences being compared differ by conservative amino acidsubstitutions.

As used herein, “amino acid difference” and “residue difference” referto a difference in the amino acid residue at a position of a polypeptidesequence relative to the amino acid residue at a corresponding positionin a reference sequence. In some cases, the reference sequence has ahistidine tag, but the numbering is maintained relative to theequivalent reference sequence without the histidine tag. The positionsof amino acid differences generally are referred to herein as “Xn,”where n refers to the corresponding position in the reference sequenceupon which the residue difference is based. For example, a “residuedifference at position X93 as compared to SEQ ID NO: 4” refers to adifference of the amino acid residue at the polypeptide positioncorresponding to position 93 of SEQ ID NO: 4. Thus, if the referencepolypeptide of SEQ ID NO: 4 has a serine at position 93, then a “residuedifference at position X93 as compared to SEQ ID NO: 4” an amino acidsubstitution of any residue other than serine at the position of thepolypeptide corresponding to position 93 of SEQ ID NO: 4. In mostinstances herein, the specific amino acid residue difference at aposition is indicated as “XnY” where “Xn” specified the correspondingposition as described above, and “Y” is the single letter identifier ofthe amino acid found in the engineered polypeptide (i.e., the differentresidue than in the reference polypeptide). In some instances (e.g., inthe Tables presented in the Examples), the present invention alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide. Insome instances, a polypeptide of the present invention can include oneor more amino acid residue differences relative to a reference sequence,which is indicated by a list of the specified positions where residuedifferences are present relative to the reference sequence. In someembodiments, where more than one amino acid can be used in a specificresidue position of a polypeptide, the various amino acid residues thatcan be used are separated by a “I” (e.g., X307H/X307P or X307H/P). Theslash may also be used to indicate multiple substitutions within a givenvariant (i.e., there is more than one substitution present in a givensequence, such as in a combinatorial variant). In some embodiments, thepresent invention includes engineered polypeptide sequences comprisingone or more amino acid differences comprising conservative ornon-conservative amino acid substitutions. In some additionalembodiments, the present invention provides engineered polypeptidesequences comprising both conservative and non-conservative amino acidsubstitutions.

As used herein, “conservative amino acid substitution” refers to asubstitution of a residue with a different residue having a similar sidechain, and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. By way of example and not limitation, in some embodiments,an amino acid with an aliphatic side chain is substituted with anotheraliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine);an amino acid with an hydroxyl side chain is substituted with anotheramino acid with an hydroxyl side chain (e.g., serine and threonine); anamino acid having an aromatic side chain is substituted with anotheramino acid having an aromatic side chain (e.g., phenylalanine, tyrosine,tryptophan, and histidine); an amino acid with a basic side chain issubstituted with another amino acid with a basic side chain (e.g.,lysine and arginine); an amino acid with an acidic side chain issubstituted with another amino acid with an acidic side chain (e.g.,aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively.

As used herein, “non-conservative substitution” refers to substitutionof an amino acid in the polypeptide with an amino acid withsignificantly differing side chain properties. Non-conservativesubstitutions may use amino acids between, rather than within, thedefined groups and affects (a) the structure of the peptide backbone inthe area of the substitution (e.g., proline for glycine) (b) the chargeor hydrophobicity, or (c) the bulk of the side chain. By way of exampleand not limitation, an exemplary non-conservative substitution can be anacidic amino acid substituted with a basic or aliphatic amino acid; anaromatic amino acid substituted with a small amino acid; and ahydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification to the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thereference enzyme while retaining enzymatic activity and/or retaining theimproved properties of an engineered peroxidase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous. Deletions are typicallyindicated by “-” in amino acid sequences.

As used herein, “insertion” refers to modification to the polypeptide byaddition of one or more amino acids from the reference polypeptide.Insertions can be in the internal portions of the polypeptide, or to thecarboxy or amino terminus. Insertions as used herein include fusionproteins as is known in the art. The insertion can be a contiguoussegment of amino acids or separated by one or more of the amino acids inthe naturally occurring polypeptide.

The term “amino acid substitution set” or “substitution set” refers to agroup of amino acid substitutions in a polypeptide sequence, as comparedto a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. Insome embodiments, a substitution set refers to the set of amino acidsubstitutions that is present in any of the variant peroxidases listedin the Tables provided in the Examples

A “functional fragment” and “biologically active fragment” are usedinterchangeably herein to refer to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion(s) and/or internaldeletions, but where the remaining amino acid sequence is identical tothe corresponding positions in the sequence to which it is beingcompared (e.g., a full-length engineered peroxidase of the presentinvention) and that retains substantially all of the activity of thefull-length polypeptide.

As used herein, “isolated polypeptide” refers to a polypeptide which issubstantially separated from other contaminants that naturally accompanyit (e.g., protein, lipids, and polynucleotides). The term embracespolypeptides which have been removed or purified from theirnaturally-occurring environment or expression system (e.g., within ahost cell or via in vitro synthesis). The recombinant peroxidasepolypeptides may be present within a cell, present in the cellularmedium, or prepared in various forms, such as lysates or isolatedpreparations. As such, in some embodiments, the recombinant peroxidasepolypeptides can be an isolated polypeptide.

As used herein, “substantially pure polypeptide” or “purified protein”refers to a composition in which the polypeptide species is thepredominant species present (i.e., on a molar or weight basis it is moreabundant than any other individual macromolecular species in thecomposition), and is generally a substantially purified composition whenthe object species comprises at least about 50 percent of themacromolecular species present by mole or % weight. However, in someembodiments, the composition comprising peroxidase comprises peroxidasethat is less than 50% pure (e.g., about 10%, about 20%, about 30%, about40%, or about 50%). Generally, a substantially pure peroxidasecomposition comprises about 60% or more, about 70% or more, about 80% ormore, about 90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedrecombinant peroxidase polypeptides are substantially pure polypeptidecompositions.

As used herein, “improved enzyme property” refers to at least oneimproved property of an enzyme. In some embodiments, the presentinvention provides engineered peroxidase polypeptides that exhibit animprovement in any enzyme property as compared to a reference peroxidasepolypeptide and/or a wild-type peroxidase polypeptide, and/or anotherengineered peroxidase polypeptide. Thus, the level of “improvement” canbe determined and compared between various peroxidase polypeptides,including wild-type, as well as engineered peroxidases. Improvedproperties include, but are not limited to, such properties as increasedprotein expression, increased thermoactivity, increased thermostability,increased pH activity, increased stability, increased enzymaticactivity, increased substrate specificity or affinity, increasedspecific activity, increased resistance to substrate or end-productinhibition, increased chemical stability, improved chemoselectivity,improved solvent stability, increased tolerance to acidic pH, increasedtolerance to proteolytic activity (i.e., reduced sensitivity toproteolysis), reduced aggregation, increased solubility, and alteredtemperature profile. In additional embodiments, the term is used inreference to the at least one improved property of peroxidase enzymes.In some embodiments, the present invention provides engineeredperoxidase polypeptides that exhibit an improvement in any enzymeproperty as compared to a reference peroxidase polypeptide and/or awild-type peroxidase polypeptide, and/or another engineered peroxidasepolypeptide. Thus, the level of “improvement” can be determined andcompared between various peroxidase polypeptides, including wild-type,as well as engineered peroxidases.

As used herein, “increased enzymatic activity” and “enhanced catalyticactivity” refer to an improved property of the engineered polypeptides,which can be represented by an increase in specific activity (e.g.,product produced/time/weight protein) or an increase in percentconversion of the substrate to the product (e.g., percent conversion ofstarting amount of substrate to product in a specified time period usinga specified amount of enzyme) as compared to the reference enzyme. Insome embodiments, the terms refer to an improved property of engineeredperoxidase polypeptides provided herein, which can be represented by anincrease in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount ofperoxidase) as compared to the reference peroxidase enzyme. In someembodiments, the terms are used in reference to improved peroxidaseenzymes provided herein. Exemplary methods to determine enzyme activityof the engineered peroxidases of the present invention are provided inthe Examples. Any property relating to enzyme activity may be affected,including the classical enzyme properties of K_(m), V_(max) or k_(cat),changes of which can lead to increased enzymatic activity. For example,improvements in enzyme activity can be from about 1.1 fold the enzymaticactivity of the corresponding wild-type enzyme, to as much as 2-fold,5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold,200-fold or more enzymatic activity than the naturally occurringperoxidase or another engineered peroxidase from which the peroxidasepolypeptides were derived.

As used herein, “conversion” refers to the enzymatic conversion (orbiotransformation) of a substrate(s) to the corresponding product(s).“Percent conversion” refers to the percent of the substrate that isconverted to the product within a period of time under specifiedconditions. Thus, the “enzymatic activity” or “activity” of a peroxidasepolypeptide can be expressed as “percent conversion” of the substrate tothe product in a specific period of time.

Enzymes with “generalist properties” (or “generalist enzymes”) refer toenzymes that exhibit improved activity for a wide range of substrates,as compared to a parental sequence. Generalist enzymes do notnecessarily demonstrate improved activity for every possible substrate.In some embodiments, the present invention provides peroxidase variantswith generalist properties, in that they demonstrate similar or improvedactivity relative to the parental gene for a wide range of stericallyand electronically diverse substrates. In addition, the generalistenzymes provided herein were engineered to be improved across a widerange of diverse molecules to increase the production ofmetabolites/products.

The term “stringent hybridization conditions” is used herein to refer toconditions under which nucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of ion strength, temperature, G/C content, andthe presence of chaotropic agents. The T_(m) values for polynucleotidescan be calculated using known methods for predicting meltingtemperatures (See e.g., Baldino et al., Meth. Enzymol., 168:761-777[1989]; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390 [1962];Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897 [1986];Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377 [1986]; Kierzeket al., Biochem., 25:7840-7846 [1986]; Rychlik et al., Nucl. Acids Res.,18:6409-6412 [1990] (erratum, Nucl. Acids Res., 19:698 [1991]); Sambrooket al., supra); Suggs et al., 1981, in Developmental Biology UsingPurified Genes, Brown et al. [eds.], pp. 683-693, Academic Press,Cambridge, Mass. [1981]; and Wetmur, Crit. Rev. Biochem. Mol. Biol.26:227-259 [1991]). In some embodiments, the polynucleotide encodes thepolypeptide disclosed herein and hybridizes under defined conditions,such as moderately stringent or highly stringent conditions, to thecomplement of a sequence encoding an engineered peroxidase enzyme of thepresent invention.

As used herein, “hybridization stringency” relates to hybridizationconditions, such as washing conditions, in the hybridization of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w/v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

As used herein, “codon optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is efficientlyexpressed in the organism of interest. Although the genetic code isdegenerate in that most amino acids are represented by several codons,called “synonyms” or “synonymous” codons, it is well known that codonusage by particular organisms is nonrandom and biased towards particularcodon triplets. This codon usage bias may be higher in reference to agiven gene, genes of common function or ancestral origin, highlyexpressed proteins versus low copy number proteins, and the aggregateprotein coding regions of an organism's genome. In some embodiments, thepolynucleotides encoding the peroxidase enzymes may be codon optimizedfor optimal production in the host organism selected for expression.

As used herein, “preferred,” “optimal,” and “high codon usage bias”codons when used alone or in combination refer(s) interchangeably tocodons that are used at higher frequency in the protein coding regionsthan other codons that code for the same amino acid. The preferredcodons may be determined in relation to codon usage in a single gene, aset of genes of common function or origin, highly expressed genes, thecodon frequency in the aggregate protein coding regions of the wholeorganism, codon frequency in the aggregate protein coding regions ofrelated organisms, or combinations thereof. Codons whose frequencyincreases with the level of gene expression are typically optimal codonsfor expression. A variety of methods are known for determining the codonfrequency (e.g., codon usage, relative synonymous codon usage) and codonpreference in specific organisms, including multivariate analysis, forexample, using cluster analysis or correspondence analysis, and theeffective number of codons used in a gene (See e.g., GCGCodonPreference, Genetics Computer Group Wisconsin Package; CodonW,Peden, University of Nottingham; McInerney, Bioinform., 14:372-73[1998]; Stenico et al., Nucl. Acids Res., 222437-46 [1994]; and Wright,Gene 87:23-29 [1990]). Codon usage tables are available for manydifferent organisms (See e.g., Wada et al., Nucl. Acids Res.,20:2111-2118 [1992]; Nakamura et al., Nucl. Acids Res., 28:292 [2000];Duret, et al., supra; Henaut and Danchin, in Escherichia coli andSalmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., p.2047-2066 [1996]). The data source for obtaining codon usage may rely onany available nucleotide sequence capable of coding for a protein. Thesedata sets include nucleic acid sequences actually known to encodeexpressed proteins (e.g., complete protein coding sequences-CDS),expressed sequence tags (ESTS), or predicted coding regions of genomicsequences (See e.g., Mount, Bioinformatics: Sequence and GenomeAnalysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [2001]; Uberbacher, Meth. Enzymol., 266:259-281 [1996]; andTiwari et al., Comput. Appl. Biosci., 13:263-270 [1997]).

As used herein, “control sequence” includes all components, which arenecessary or advantageous for the expression of a polynucleotide and/orpolypeptide of the present invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter sequence, signalpeptide sequence, initiation sequence and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

The phrase “suitable reaction conditions” refers to those conditions inthe enzymatic conversion reaction solution (e.g., ranges of enzymeloading, substrate loading, temperature, pH, buffers, co-solvents, etc.)under which a peroxidase polypeptide of the present invention is capableof converting a substrate to the desired product compound. Someexemplary “suitable reaction conditions” are provided herein and/or inthe Examples.

As used herein, “loading,” such as in “compound loading” or “enzymeloading” refers to the concentration or amount of a component in areaction mixture at the start of the reaction.

As used herein, “substrate” in the context of an enzymatic conversionreaction process refers to the compound or molecule acted on by theengineered enzymes provided herein (e.g., engineered peroxidasepolypeptides).

As used herein, “increasing” yield of a product (e.g.,7-hydroxy-3H-phenoxazin-3-one (Resorufin)) from a reaction occurs when aparticular component present during the reaction (e.g., a peroxidaseenzyme) causes more product to be produced, compared with a reactionconducted under the same conditions with the same substrate and othersubstituents, but in the absence of the component of interest.

A reaction is said to be “substantially free” of a particular enzyme ifthe amount of that enzyme compared with other enzymes that participatein catalyzing the reaction is less than about 2%, about 1%, or about0.1% (wt/wt).

As used herein, “fractionating” a liquid (e.g., a culture broth) meansapplying a separation process (e.g., salt precipitation, columnchromatography, size exclusion, and filtration) or a combination of suchprocesses to provide a solution in which a desired protein comprises agreater percentage of total protein in the solution than in the initialliquid product.

As used herein, “starting composition” refers to any composition thatcomprises at least one substrate. In some embodiments, the startingcomposition comprises any suitable substrate.

As used herein, “product” in the context of an enzymatic conversionprocess refers to the compound or molecule resulting from the action ofan enzymatic polypeptide on a substrate.

As used herein, “equilibration” as used herein refers to the processresulting in a steady state concentration of chemical species in achemical or enzymatic reaction (e.g., interconversion of two species Aand B), including interconversion of stereoisomers, as determined by theforward rate constant and the reverse rate constant of the chemical orenzymatic reaction.

“Cofactor,” as used herein, refers to a non-protein compound thatoperates in combination with an enzyme in catalyzing a reaction.

As used herein, “amino” refers to the group —NH₂.

As used herein, “oxo” refers to ═O.

As used herein, “oxy” refers to a divalent group —O—, which may havevarious substituents to form different oxy groups, including ethers andesters.

As used herein, “carboxy” refers to —COOH.

As used herein, “carbonyl” refers to —C(O)—, which may have a variety ofsubstituents to form different carbonyl groups including acids, acidhalides, aldehydes, amides, esters, and ketones.

As used herein, “hydroxy” refers to —OH.

As used herein, “heteroaryl” refers to an aromatic heterocyclic group offrom 1 to 10 carbon atoms inclusively and 1 to 4 heteroatoms inclusivelyselected from oxygen, nitrogen and sulfur within the ring. Suchheteroaryl groups can have a single ring (e.g., pyridyl or furyl) ormultiple condensed rings (e.g., indolizinyl or benzothienyl).

As used herein, “membered ring” is meant to embrace any cyclicstructure. The number preceding the term “membered” denotes the numberof skeletal atoms that constitute the ring. Thus, for example,cyclohexyl, pyridine, pyran and thiopyran are 6-membered rings andcyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

As used herein the term “culturing” refers to the growing of apopulation of microbial cells under any suitable conditions (e.g., usinga liquid, gel or solid medium).

Recombinant polypeptides can be produced using any suitable methodsknown in the art. Genes encoding the wild-type polypeptide of interestcan be cloned in vectors, such as plasmids, and expressed in desiredhosts, such as E. coli, etc. Variants of recombinant polypeptides can begenerated by various methods known in the art. Indeed, there is a widevariety of different mutagenesis techniques well known to those skilledin the art. In addition, mutagenesis kits are also available from manycommercial molecular biology suppliers. Methods are available to makespecific substitutions at defined amino acids (site-directed), specificor random mutations in a localized region of the gene (regio-specific),or random mutagenesis over the entire gene (e.g., saturationmutagenesis). Numerous suitable methods are known to those in the art togenerate enzyme variants, including but not limited to site-directedmutagenesis of single-stranded DNA or double-stranded DNA using PCR,cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, andchemical saturation mutagenesis, or any other suitable method known inthe art. Mutagenesis and directed evolution methods can be readilyapplied to enzyme-encoding polynucleotides to generate variant librariesthat can be expressed, screened, and assayed. Any suitable mutagenesisand directed evolution methods find use in the present invention and arewell known in the art (See e.g., U.S. Pat. Nos. 5,605,793, 5,811,238,5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679,6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638,6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883,6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198,6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377,6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964,6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910,6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675,6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105,6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065,6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467,6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072,6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882,6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297,7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477,7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464,7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030,7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249,7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001,8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346,8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, andall related US, as well as PCT and non-US counterparts; Ling et al.,Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol.,57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botsteinet al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7[1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene,34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290[1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameriet al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol.,15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319[1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad.Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966;WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of whichare incorporated herein by reference).

In some embodiments, the enzyme clones obtained following mutagenesistreatment are screened by subjecting the enzyme preparations to adefined temperature (or other assay conditions) and measuring the amountof enzyme activity remaining after heat treatments or other suitableassay conditions. Clones containing a polynucleotide encoding apolypeptide are then isolated from the gene, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell. Measuring enzyme activity from the expression libraries canbe performed using any suitable method known in the art (e.g., standardbiochemistry techniques, such as HPLC analysis).

After the variants are produced, they can be screened for any desiredproperty (e.g., high or increased activity, or low or reduced activity,increased thermal activity, increased thermal stability, and/or acidicpH stability, etc.). In some embodiments, “recombinant peroxidasepolypeptides” (also referred to herein as “engineered peroxidasepolypeptides,” “variant peroxidase enzymes,” “peroxidase variants,” and“peroxidase combinatorial variants”) find use. In some embodiments,“recombinant peroxidase polypeptides” (also referred to as “engineeredperoxidase polypeptides,” “variant peroxidase enzymes,” “peroxidasevariants,” and “peroxidase combinatorial variants”) find use.

As used herein, a “vector” is a DNA construct for introducing a DNAsequence into a cell. In some embodiments, the vector is an expressionvector that is operably linked to a suitable control sequence capable ofeffecting the expression in a suitable host of the polypeptide encodedin the DNA sequence. In some embodiments, an “expression vector” has apromoter sequence operably linked to the DNA sequence (e.g., transgene)to drive expression in a host cell, and in some embodiments, alsocomprises a transcription terminator sequence.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation, andpost-translational modification. In some embodiments, the term alsoencompasses secretion of the polypeptide from a cell.

As used herein, the term “produces” refers to the production of proteinsand/or other compounds by cells. It is intended that the term encompassany step involved in the production of polypeptides including, but notlimited to, transcription, post-transcriptional modification,translation, and post-translational modification. In some embodiments,the term also encompasses secretion of the polypeptide from a cell.

As used herein, an amino acid or nucleotide sequence (e.g., a promotersequence, signal peptide, terminator sequence, etc.) is “heterologous”to another sequence with which it is operably linked if the twosequences are not associated in nature. For example a “heterologouspolynucleotide” is any polynucleotide that is introduced into a hostcell by laboratory techniques, and includes polynucleotides that areremoved from a host cell, subjected to laboratory manipulation, and thenreintroduced into a host cell.

As used herein, the terms “host cell” and “host strain” refer tosuitable hosts for expression vectors comprising DNA provided herein(e.g., the polynucleotides encoding the peroxidase variants). In someembodiments, the host cells are prokaryotic or eukaryotic cells thathave been transformed or transfected with vectors constructed usingrecombinant DNA techniques as known in the art.

The term “analogue” means a polypeptide having more than 70% sequenceidentity but less than 100% sequence identity (e.g., more than 75%, 78%,80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity) with a reference polypeptide. In some embodiments,analogues means polypeptides that contain one or more non-naturallyoccurring amino acid residues including, but not limited, tohomoarginine, ornithine and norvaline, as well as naturally occurringamino acids. In some embodiments, analogues also include one or moreD-amino acid residues and non-peptide linkages between two or more aminoacid residues.

The term “effective amount” means an amount sufficient to produce thedesired result. One of general skill in the art may determine theeffective amount by using routine experimentation.

The terms “isolated” and “purified” are used to refer to a molecule(e.g., an isolated nucleic acid, polypeptide, etc.) or other componentthat is removed from at least one other component with which it isnaturally associated. The term “purified” does not require absolutepurity, rather it is intended as a relative definition.

As used herein, “stereoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (“e.e.”) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (“d.e.”).Enantiomeric excess and diastereomeric excess are types of stereomericexcess.

As used herein, “regioselectivity” and “regioselective reaction” referto a reaction in which one direction of bond making or breaking occurspreferentially over all other possible directions. Reactions cancompletely (100%) regioselective if the discrimination is complete,substantially regioselective (at least 75%), or partially regioselective(x %, wherein the percentage is set dependent upon the reaction ofinterest), if the product of reaction at one site predominates over theproduct of reaction at other sites.

As used herein, “chemoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one product over another.

As used herein, “pH stable” refers to a peroxidase polypeptide thatmaintains similar activity (e.g., more than 60% to 80%) after exposureto high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme.

As used herein, “thermostable” refers to a peroxidase polypeptide thatmaintains similar activity (more than 60% to 80% for example) afterexposure to elevated temperatures (e.g., 40-80° C.) for a period of time(e.g., 0.5-24 h) compared to the wild-type enzyme exposed to the sameelevated temperature.

As used herein, “solvent stable” refers to a peroxidase polypeptide thatmaintains similar activity (more than e.g., 60% to 80%) after exposureto varying concentrations (e.g., 5-99%) of solvent (ethanol, isopropylalcohol, dimethylsulfoxide [DMSO], tetrahydrofuran,2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyltert-butyl ether, etc.) for a period of time (e.g., 0.5-24 h) comparedto the wild-type enzyme exposed to the same concentration of the samesolvent.

As used herein, “thermo- and solvent stable” refers to a peroxidasepolypeptide that is both thermostable and solvent stable.

As used herein, “optional” and “optionally” mean that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. One of ordinary skill in the artwould understand that with respect to any molecule described ascontaining one or more optional substituents, only sterically practicaland/or synthetically feasible compounds are meant to be included.

As used herein, “optionally substituted” refers to all subsequentmodifiers in a term or series of chemical groups. For example, in theterm “optionally substituted arylalkyl, the “alkyl” portion and the“aryl” portion of the molecule may or may not be substituted, and forthe series “optionally substituted alkyl, cycloalkyl, aryl andheteroaryl,” the alkyl, cycloalkyl, aryl, and heteroaryl groups,independently of the others, may or may not be substituted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides engineered peroxidase enzymes,polypeptides having peroxidase activity, and polynucleotides encodingthese enzymes, as well as vectors and host cells comprising thesepolynucleotides and polypeptides. Methods for producing peroxidaseenzymes are also provided. The present invention further providescompositions comprising the peroxidase enzymes and methods of using theengineered peroxidase enzymes. The present invention finds particularuse in the production of pharmaceutical and other compounds.

DyP-type peroxidase (DyP) (EC number 1.11.1.19) is a family of dyede-colorizing peroxidase enzymes. The enzyme is a haem containingprotein that catalyzes the oxidation of a number of substrates usinghydrogen peroxide as the electron donor. The engineered DyP peroxidaseused in this method has a sequence derived from the sequence of thewild-type peroxidase Alr1585 from cyanobacteria Anabaena (Nostoc) sp.(strain PCC 7120). The DyP peroxidase catalyzes the production of7-hydroxy-3H-phenoxazin-3-one (Resorufin) using10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) as the substrate asdepicted in Scheme 1, below.

Engineered Peroxidase Polypeptides

The present invention provides engineered peroxidase polypeptides,polynucleotides encoding the polypeptides, methods of preparing thepolypeptides, and methods for using the polypeptides. Where thedescription relates to polypeptides, it is to be understood that it alsodescribes the polynucleotides encoding the polypeptides. In someembodiments, the present invention provides engineered, non-naturallyoccurring peroxidase enzymes with improved properties as compared towild-type peroxidase enzymes.

In some embodiments, the engineered peroxidase polypeptides can be usedfor the conversion of an organic substrate using hydrogen peroxide asthe electron donor. In some embodiments, the engineered peroxidasepolypeptides can be used for the conversion of the substrate compound(1) (10-acetyl-3,7-dihydroxyphenoxazine or Amplex Red)

to the product compound (2) (7-hydroxy-3H-phenoxazin-3-one or Resorufin)

In some embodiments, the engineered peroxidase polypeptides can be usedto promote oxidase catalytic activity by oxidizing an inactive redoxstate of the active site during normal oxidase catalytic cycling.

Any suitable reaction conditions find use in the present invention. Insome embodiments, methods are used to analyze the improved properties ofthe engineered polypeptides to carry out the oxidation reaction. In someembodiments, the reaction conditions are modified with regard toconcentrations or amounts of engineered peroxidase, substrate(s),buffer(s), solvent(s), co-factors, pH, conditions including temperatureand reaction time, and/or conditions with the engineered peroxidasepolypeptide immobilized on a solid support, as further described belowand in the Examples.

In some embodiments, the suitable reaction conditions comprise: (a)substrate loading at about 1 g/L to 50 g/L; (b) about 0.1 g/L to 10 g/Lof the engineered polypeptide; (c) about 0.5 mM to 2.5 mM hydrogenperoxide; (d) a pH of about 6 to 7; (e) temperature of about 20° to 40°C.; and (h) reaction time of 2-30 mins.

In some embodiments, the suitable reaction conditions comprise: (a)substrate loading at about 13 g/L; (b) about 5 g/L of the engineeredpolypeptide; (c) about 1.3 mM hydrogen peroxide; (d) a pH of about 6.5;(g) temperature of about 23° C.; and (h) reaction time of 2 mins.

In some embodiments, additional reaction components or additionaltechniques are utilized to supplement the reaction conditions. In someembodiments, these include taking measures to stabilize or preventinactivation of the enzyme, reduce product inhibition, and/or shiftreaction equilibrium to desired product formation.

In some further embodiments, any of the above described processes forthe conversion of substrate compound to product compound can furthercomprise one or more steps selected from: extraction, isolation,purification, crystallization, filtration, and/or lyophilization ofproduct compound(s). Methods, techniques, and protocols for extracting,isolating, purifying, and/or crystallizing the product(s) frombiocatalytic reaction mixtures produced by the processes provided hereinare known to the ordinary artisan and/or accessed through routineexperimentation. Additionally, illustrative methods are provided in theExamples below.

Engineered Peroxidase Polynucleotides Encoding Engineered Polypeptides,Expression Vectors and Host Cells

The present invention provides polynucleotides encoding the engineeredenzyme polypeptides described herein. In some embodiments, thepolynucleotides are operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide. Insome embodiments, expression constructs containing at least oneheterologous polynucleotide encoding the engineered enzymepolypeptide(s) are introduced into appropriate host cells to express thecorresponding enzyme polypeptide(s).

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode an engineered enzyme (e.g., peroxidase) polypeptide.Thus, the present invention provides methods and compositions for theproduction of each and every possible variation of enzymepolynucleotides that could be made that encode the enzyme polypeptidesdescribed herein by selecting combinations based on the possible codonchoices, and all such variations are to be considered specificallydisclosed for any polypeptide described herein, including the amino acidsequences presented in the Examples (e.g., in the various Tables).

In some embodiments, the codons are preferably optimized for utilizationby the chosen host cell for protein production. For example, preferredcodons used in bacteria are typically used for expression in bacteria.Consequently, codon optimized polynucleotides encoding the engineeredenzyme polypeptides contain preferred codons at about 40%, 50%, 60%,70%, 80%, 90%, or greater than 90% of the codon positions in the fulllength coding region.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding the naturally occurring peroxidase polypeptide amino acidsequence, as represented by SEQ ID NO: 1. In some embodiments, thepolynucleotide has a nucleic acid sequence comprising at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to thenucleic acid sequences of SEQ ID NO: 3 or 5, each of which encodes theidentical polypeptide sequences of SEQ ID NO: 4 or 6, respectively.

In some embodiments, the enzyme polynucleotide encodes an engineeredpolypeptide having enzyme activity with the properties disclosed herein,wherein the polypeptide comprises an amino acid sequence having at least60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequenceselected from the SEQ ID NOS provided herein, or the amino acid sequenceof any variant (e.g., those provided in the Examples), and one or moreresidue differences as compared to the reference polynucleotide(s), orthe amino acid sequence of any variant as disclosed in the Examples (forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residuepositions). In some embodiments, the reference polypeptide sequence isselected from SEQ ID NOS: 4 and 6.

In some embodiments, the polynucleotide encoding the engineeredperoxidase comprises a polynucleotide sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identity to a sequence selected from SEQ IDNOS: 3 and/or 5. In some embodiments, the polynucleotide encoding theengineered peroxidase comprises SEQ ID NO: 5. In some embodiments, thepolynucleotide encoding the engineered peroxidase comprises apolynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identity to a sequence selected from SEQ ID NOS: 3 to 289. In someembodiments, the polynucleotide encoding the engineered peroxidasecomprises a polynucleotide sequence selected from SEQ ID NOS: 5 to 289.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from any polynucleotide sequence provided herein, or acomplement thereof, or a polynucleotide sequence encoding any of thevariant enzyme polypeptides provided herein. In some embodiments, thepolynucleotide capable of hybridizing under highly stringent conditionsencodes an enzyme polypeptide comprising an amino acid sequence that hasone or more residue differences as compared to a reference sequence.

In some embodiments, an isolated polynucleotide encoding any of theengineered enzyme polypeptides herein is manipulated in a variety ofways to facilitate expression of the enzyme polypeptide. In someembodiments, the polynucleotides encoding the enzyme polypeptidescomprise expression vectors where one or more control sequences ispresent to regulate the expression of the enzyme polynucleotides and/orpolypeptides. Manipulation of the isolated polynucleotide prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector utilized. Techniques for modifying polynucleotides andnucleic acid sequences utilizing recombinant DNA methods are well knownin the art. In some embodiments, the control sequences include amongothers, promoters, leader sequences, polyadenylation sequences,propeptide sequences, signal peptide sequences, and transcriptionterminators. In some embodiments, suitable promoters are selected basedon the host cells selection. For bacterial host cells, suitablepromoters for directing transcription of the nucleic acid constructs ofthe present disclosure, include, but are not limited to promotersobtained from the E. coli lac operon, Streptomyces coelicolor agarasegene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacilluslicheniformis alpha-amylase gene (amyL), Bacillus stearothermophilusmaltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylasegene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillussubtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Seee.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731[1978]), as well as the tac promoter (See e.g., DeBoer et al., Proc.Natl Acad. Sci. USA 80: 21-25 [1983]). Exemplary promoters forfilamentous fungal host cells, include, but are not limited to promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusariumoxysporum trypsin-like protease (See e.g., WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof. Exemplary yeast cell promoters can be from the genes can befrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.Other useful promoters for yeast host cells are known in the art (Seee.g., Romanos et al., Yeast 8:423-488 [1992]).

In some embodiments, the control sequence is also a suitabletranscription terminator sequence (i.e., a sequence recognized by a hostcell to terminate transcription). In some embodiments, the terminatorsequence is operably linked to the 3′ terminus of the nucleic acidsequence encoding the enzyme polypeptide. Any suitable terminator whichis functional in the host cell of choice finds use in the presentinvention. Exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease. Exemplary terminators for yeast hostcells can be obtained from the genes for Saccharomyces cerevisiaeenolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomycescerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other usefulterminators for yeast host cells are known in the art (See e.g., Romanoset al., supra).

In some embodiments, the control sequence is also a suitable leadersequence (i.e., a non-translated region of an mRNA that is important fortranslation by the host cell). In some embodiments, the leader sequenceis operably linked to the 5′ terminus of the nucleic acid sequenceencoding the enzyme polypeptide. Any suitable leader sequence that isfunctional in the host cell of choice find use in the present invention.Exemplary leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cells areobtained from the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

In some embodiments, the control sequence is also a polyadenylationsequence (i.e., a sequence operably linked to the 3′ terminus of thenucleic acid sequence and which, when transcribed, is recognized by thehost cell as a signal to add polyadenosine residues to transcribedmRNA). Any suitable polyadenylation sequence which is functional in thehost cell of choice finds use in the present invention. Exemplarypolyadenylation sequences for filamentous fungal host cells include, butare not limited to the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase. Useful polyadenylation sequences for yeast hostcells are known (See e.g., Guo and Sherman, Mol. Cell. Biol.,15:5983-5990 [1995]).

In some embodiments, the control sequence is also a signal peptide(i.e., a coding region that codes for an amino acid sequence linked tothe amino terminus of a polypeptide and directs the encoded polypeptideinto the cell's secretory pathway). In some embodiments, the 5′ end ofthe coding sequence of the nucleic acid sequence inherently contains asignal peptide coding region naturally linked in translation readingframe with the segment of the coding region that encodes the secretedpolypeptide. Alternatively, in some embodiments, the 5′ end of thecoding sequence contains a signal peptide coding region that is foreignto the coding sequence. Any suitable signal peptide coding region whichdirects the expressed polypeptide into the secretory pathway of a hostcell of choice finds use for expression of the engineeredpolypeptide(s). Effective signal peptide coding regions for bacterialhost cells are the signal peptide coding regions include, but are notlimited to those obtained from the genes for Bacillus NClB 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are known in the art(See e.g., Simonen and Palva, Microbiol. Rev., 57:109-137 [1993]). Insome embodiments, effective signal peptide coding regions forfilamentous fungal host cells include, but are not limited to the signalpeptide coding regions obtained from the genes for Aspergillus oryzaeTAKA amylase, Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolenscellulase, and Humicola lanuginosa lipase. Useful signal peptides foryeast host cells include, but are not limited to those from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase.

In some embodiments, the control sequence is also a propeptide codingregion that codes for an amino acid sequence positioned at the aminoterminus of a polypeptide. The resultant polypeptide is referred to as a“proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from any suitable source, including, butnot limited to the genes for Bacillus subtilis alkaline protease (aprE),Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiaealpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthorathermophila lactase (See e.g., WO 95/33836). Where both signal peptideand propeptide regions are present at the amino terminus of apolypeptide, the propeptide region is positioned next to the aminoterminus of a polypeptide and the signal peptide region is positionednext to the amino terminus of the propeptide region.

In some embodiments, regulatory sequences are also utilized. Thesesequences facilitate the regulation of the expression of the polypeptiderelative to the growth of the host cell. Examples of regulatory systemsare those that cause the expression of the gene to be turned on or offin response to a chemical or physical stimulus, including the presenceof a regulatory compound. In prokaryotic host cells, suitable regulatorysequences include, but are not limited to the lac, tac, and trp operatorsystems. In yeast host cells, suitable regulatory systems include, butare not limited to the ADH2 system or GAL1 system. In filamentous fungi,suitable regulatory sequences include, but are not limited to the TAKAalpha-amylase promoter, Aspergillus niger glucoamylase promoter, andAspergillus oryzae glucoamylase promoter.

In another aspect, the present invention is directed to a recombinantexpression vector comprising a polynucleotide encoding an engineeredenzyme polypeptide, and one or more expression regulating regions suchas a promoter and a terminator, a replication origin, etc., depending onthe type of hosts into which they are to be introduced. In someembodiments, the various nucleic acid and control sequences describedherein are joined together to produce recombinant expression vectorswhich include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding theenzyme polypeptide at such sites. Alternatively, in some embodiments,the nucleic acid sequence of the present invention is expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Insome embodiments involving the creation of the expression vector, thecoding sequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any suitable vector (e.g., aplasmid or virus), that can be conveniently subjected to recombinant DNAprocedures and bring about the expression of the enzyme polynucleotidesequence. The choice of the vector typically depends on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extra-chromosomal element, aminichromosome, or an artificial chromosome). The vector may contain anymeans for assuring self-replication. In some alternative embodiments,the vector is one in which, when introduced into the host cell, it isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, in someembodiments, a single vector or plasmid, or two or more vectors orplasmids which together contain the total DNA to be introduced into thegenome of the host cell, and/or a transposon is utilized.

In some embodiments, the expression vector contains one or moreselectable markers, which permit easy selection of transformed cells. A“selectable marker” is a gene, the product of which provides for biocideor viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markersinclude, but are not limited to the dal genes from Bacillus subtilis orBacillus licheniformis, or markers, which confer antibiotic resistancesuch as ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Suitable markers for yeast host cells include, but are notlimited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in filamentous fungal host cells include, but are notlimited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae),argB (ornithine carbamoyltransferases), bar (phosphinothricinacetyltransferase; e.g., from S. hygroscopicus), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase; e.g., from A. nidulans or A.orzyae), sC (sulfate adenyltransferase), and trpC (anthranilatesynthase), as well as equivalents thereof.

In another aspect, the present invention provides a host cell comprisingat least one polynucleotide encoding at least one engineered enzymepolypeptide of the present invention, the polynucleotide(s) beingoperatively linked to one or more control sequences for expression ofthe engineered enzyme enzyme(s) in the host cell. Host cells suitablefor use in expressing the polypeptides encoded by the expression vectorsof the present invention are well known in the art and include but arenot limited to, bacterial cells, such as E. coli, Vibrio fluvialis,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. Exemplary host cells also includevarious Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21).Examples of bacterial selectable markers include, but are not limited tothe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol, and or tetracycline resistance.

In some embodiments, the expression vectors of the present inventioncontain an element(s) that permits integration of the vector into thehost cell's genome or autonomous replication of the vector in the cellindependent of the genome. In some embodiments involving integrationinto the host cell genome, the vectors rely on the nucleic acid sequenceencoding the polypeptide or any other element of the vector forintegration of the vector into the genome by homologous or nonhomologousrecombination.

In some alternative embodiments, the expression vectors containadditional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements preferably contain a sufficient number ofnucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000base pairs, and most preferably 800 to 10,000 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, or pTA1060 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes it's functioningtemperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl.Acad. Sci. USA 75:1433 [1978]).

In some embodiments, more than one copy of a nucleic acid sequence ofthe present invention is inserted into the host cell to increaseproduction of the gene product. An increase in the copy number of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the nucleic acidsequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the nucleic acid sequence,can be selected for by cultivating the cells in the presence of theappropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors include,but are not limited to the p3xFLAGTM™ expression vectors (Sigma-AldrichChemicals), which include a CMV promoter and hGH polyadenylation sitefor expression in mammalian host cells and a pBR322 origin ofreplication and ampicillin resistance markers for amplification in E.coli. Other suitable expression vectors include, but are not limited topBluescriptII SK(−) and pBK-CMV (Stratagene), and plasmids derived frompBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly(See e.g., Lathe et al., Gene 57:193-201 [1987]).

Thus, in some embodiments, a vector comprising a sequence encoding atleast one variant peroxidase is transformed into a host cell in order toallow propagation of the vector and expression of the variantperoxidase(s). In some embodiments, the variant peroxidases arepost-translationally modified to remove the signal peptide, and in somecases, may be cleaved after secretion. In some embodiments, thetransformed host cell described above is cultured in a suitable nutrientmedium under conditions permitting the expression of the variantperoxidase(s). Any suitable medium useful for culturing the host cellsfinds use in the present invention, including, but not limited tominimal or complex media containing appropriate supplements. In someembodiments, host cells are grown in HTP media. Suitable media areavailable from various commercial suppliers or may be prepared accordingto published recipes (e.g., in catalogues of the American Type CultureCollection).

In another aspect, the present invention provides host cells comprisinga polynucleotide encoding an improved peroxidase polypeptide providedherein, the polynucleotide being operatively linked to one or morecontrol sequences for expression of the peroxidase enzyme in the hostcell. Host cells for use in expressing the peroxidase polypeptidesencoded by the expression vectors of the present invention are wellknown in the art and include but are not limited to, bacterial cells,such as E. coli, Bacillus megaterium, Lactobacillus kefir, Streptomycesand Salmonella typhimurium cells; fungal cells, such as yeast cells(e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; andplant cells. Appropriate culture media and growth conditions for theabove-described host cells are well known in the art.

Polynucleotides for expression of the peroxidase may be introduced intocells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells areknown to those skilled in the art.

In some embodiments, the host cell is a eukaryotic cell. Suitableeukaryotic host cells include, but are not limited to, fungal cells,algal cells, insect cells, and plant cells. Suitable fungal host cellsinclude, but are not limited to, Ascomycota, Basidiomycota,Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, thefungal host cells are yeast cells and filamentous fungal cells. Thefilamentous fungal host cells of the present invention include allfilamentous forms of the subdivision Eumycotina and Oomycota.Filamentous fungi are characterized by a vegetative mycelium with a cellwall composed of chitin, cellulose and other complex polysaccharides.The filamentous fungal host cells of the present invention aremorphologically distinct from yeast.

In some embodiments of the present invention, the filamentous fungalhost cells are of any suitable genus and species, including, but notlimited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus,Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis,Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora,Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces,Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium,Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes,Tolypocladium, Trichoderma, Verticillium, and/or Volvariella, and/orteleomorphs, or anamorphs, and synonyms, basionyms, or taxonomicequivalents thereof.

In some embodiments of the present invention, the host cell is a yeastcell, including but not limited to cells of Candida, Hansenula,Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowiaspecies. In some embodiments of the present invention, the yeast cell isHansenula polymorpha, Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis,Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia quercuum, Pichia pijperi Pichia stipitis Pichiamethanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, orYarrowia lipolytica.

In some embodiments of the invention, the host cell is an algal cellsuch as Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp.ATCC29409).

In some other embodiments, the host cell is a prokaryotic cell. Suitableprokaryotic cells include, but are not limited to Gram-positive,Gram-negative and Gram-variable bacterial cells. Any suitable bacterialorganism finds use in the present invention, including but not limitedto Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter,Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium,Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter,Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia,Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium,Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter,Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus,Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium,Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus,Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia,Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus,Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella,Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, temecula,Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella,Yersinia and Zymomonas. In some embodiments, the host cell is a speciesof Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium,Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium,Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus,Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella,Streptococcus, Streptomyces, or Zymomonas. In some embodiments, thebacterial host strain is non-pathogenic to humans. In some embodimentsthe bacterial host strain is an industrial strain. Numerous bacterialindustrial strains are known and suitable in the present invention. Insome embodiments of the present invention, the bacterial host cell is anAgrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A.rubi). In some embodiments of the present invention, the bacterial hostcell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A.globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A.paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus, and A.ureafaciens). In some embodiments of the present invention, thebacterial host cell is a Bacillus species (e.g., B. thuringensis, B.anthracis, B. megaterium, B. subtilis, B. lentus, B. circulars, B.pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius,B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, andB. amyloliquefaciens). In some embodiments, the host cell is anindustrial Bacillus strain including but not limited to B. subtilis, B.pumilus, B. licheniformis, B. megaterium, B. clausii, B.stearothermophilus, or B. amyloliquefaciens. In some embodiments, theBacillus host cells are B. subtilis, B. licheniformis, B. megaterium, B.stearothermophilus, and/or B. amyloliquefaciens. In some embodiments,the bacterial host cell is a Clostridium species (e.g., C.acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum,C. perfringens, and C. beijerinckii). In some embodiments, the bacterialhost cell is a Corynebacterium species (e.g., C. glutamicum and C.acetoacidophilum). In some embodiments the bacterial host cell is anEscherichia species (e.g., E. coli). In some embodiments, the host cellis Escherichia coli W3110. In some embodiments, the bacterial host cellis an Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas, E.herbicola, E. punctata, and E. terreus). In some embodiments, thebacterial host cell is a Pantoea species (e.g., P. citrea, and P.agglomerans). In some embodiments the bacterial host cell is aPseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, andP. sp. D-0l 10). In some embodiments, the bacterial host cell is aStreptococcus species (e.g., S. equisimiles, S. pyogenes, and S.uberis). In some embodiments, the bacterial host cell is a Streptomycesspecies (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S.coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, andS. lividans). In some embodiments, the bacterial host cell is aZymomonas species (e.g., Z. mobilis, and Z. lipolytica).

Many prokaryotic and eukaryotic strains that find use in the presentinvention are readily available to the public from a number of culturecollections such as American Type Culture Collection (ATCC), DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSM), CentraalbureauVoor Schimmelcultures (CBS), and Agricultural Research Service PatentCulture Collection, Northern Regional Research Center (NRRL).

In some embodiments, host cells are genetically modified to havecharacteristics that improve protein secretion, protein stability and/orother properties desirable for expression and/or secretion of a protein.Genetic modification can be achieved by genetic engineering techniquesand/or classical microbiological techniques (e.g., chemical or UVmutagenesis and subsequent selection). Indeed, in some embodiments,combinations of recombinant modification and classical selectiontechniques are used to produce the host cells. Using recombinanttechnology, nucleic acid molecules can be introduced, deleted, inhibitedor modified, in a manner that results in increased yields of peroxidasevariant(s) within the host cell and/or in the culture medium. Forexample, knockout of Alp1 function results in a cell that is proteasedeficient, and knockout of pyr5 function results in a cell with apyrimidine deficient phenotype. In one genetic engineering approach,homologous recombination is used to induce targeted gene modificationsby specifically targeting a gene in vivo to suppress expression of theencoded protein. In alternative approaches, siRNA, antisense and/orribozyme technology find use in inhibiting gene expression. A variety ofmethods are known in the art for reducing expression of protein incells, including, but not limited to deletion of all or part of the geneencoding the protein and site-specific mutagenesis to disrupt expressionor activity of the gene product. (See e.g., Chaveroche et al., Nucl.Acids Res., 28:22 e97 [2000]; Cho et al., Molec. Plant MicrobeInteract., 19:7-15 [2006]; Maruyama and Kitamoto, Biotechnol Lett.,30:1811-1817 [2008]; Takahashi et al., Mol. Gen. Genom., 272: 344-352[2004]; and You et al., Arch. Microbiol., 191:615-622 [2009], all ofwhich are incorporated by reference herein). Random mutagenesis,followed by screening for desired mutations also finds use (See e.g.,Combier et al., FEMS Microbiol. Lett., 220:141-8 [2003]; and Firon etal., Eukary. Cell 2:247-55 [2003], both of which are incorporated byreference).

Introduction of a vector or DNA construct into a host cell can beaccomplished using any suitable method known in the art, including butnot limited to calcium phosphate transfection, DEAE-dextran mediatedtransfection, PEG-mediated transformation, electroporation, or othercommon techniques known in the art. In some embodiments, the Escherichiacoli expression vector pCK100900i (See, U.S. Pat. No. 9,714,437, whichis hereby incorporated by reference) finds use.

In some embodiments, the engineered host cells (i.e., “recombinant hostcells”) of the present invention are cultured in conventional nutrientmedia modified as appropriate for activating promoters, selectingtransformants, or amplifying the peroxidase polynucleotide. Cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and are well-known tothose skilled in the art. As noted, many standard references and textsare available for the culture and production of many cells, includingcells of bacterial, plant, animal (especially mammalian) andarchebacterial origin.

In some embodiments, cells expressing the variant peroxidasepolypeptides of the invention are grown under batch or continuousfermentations conditions. Classical “batch fermentation” is a closedsystem, wherein the compositions of the medium is set at the beginningof the fermentation and is not subject to artificial alternations duringthe fermentation. A variation of the batch system is a “fed-batchfermentation” which also finds use in the present invention. In thisvariation, the substrate is added in increments as the fermentationprogresses. Fed-batch systems are useful when catabolite repression islikely to inhibit the metabolism of the cells and where it is desirableto have limited amounts of substrate in the medium. Batch and fed-batchfermentations are common and well known in the art. “Continuousfermentation” is an open system where a defined fermentation medium isadded continuously to a bioreactor and an equal amount of conditionedmedium is removed simultaneously for processing. Continuous fermentationgenerally maintains the cultures at a constant high density where cellsare primarily in log phase growth. Continuous fermentation systemsstrive to maintain steady state growth conditions. Methods formodulating nutrients and growth factors for continuous fermentationprocesses as well as techniques for maximizing the rate of productformation are well known in the art of industrial microbiology.

In some embodiments of the present invention, cell-freetranscription/translation systems find use in producing variantperoxidase(s). Several systems are commercially available and themethods are well-known to those skilled in the art.

The present invention provides methods of making variant peroxidasepolypeptides or biologically active fragments thereof. In someembodiments, the method comprises: providing a host cell transformedwith a polynucleotide encoding an amino acid sequence that comprises atleast about 70% (or at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%) sequenceidentity to SEQ ID NO: 4 and/6, and comprising at least one mutation asprovided herein; culturing the transformed host cell in a culture mediumunder conditions in which the host cell expresses the encoded variantperoxidase polypeptide; and optionally recovering or isolating theexpressed variant peroxidase polypeptide, and/or recovering or isolatingthe culture medium containing the expressed variant peroxidasepolypeptide. In some embodiments, the methods further provide optionallylysing the transformed host cells after expressing the encodedperoxidase polypeptide and optionally recovering and/or isolating theexpressed variant peroxidase polypeptide from the cell lysate. Thepresent invention further provides methods of making a variantperoxidase polypeptide comprising cultivating a host cell transformedwith a variant peroxidase polypeptide under conditions suitable for theproduction of the variant peroxidase polypeptide and recovering thevariant peroxidase polypeptide. Typically, recovery or isolation of theperoxidase polypeptide is from the host cell culture medium, the hostcell or both, using protein recovery techniques that are well known inthe art, including those described herein. In some embodiments, hostcells are harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including, but not limited tofreeze-thaw cycling, sonication, mechanical disruption, and/or use ofcell lysing agents, as well as many other suitable methods well known tothose skilled in the art.

Engineered peroxidase enzymes expressed in a host cell can be recoveredfrom the cells and/or the culture medium using any one or more of thetechniques known in the art for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™(Sigma-Aldrich). Thus, in some embodiments, the resulting polypeptide isrecovered/isolated and optionally purified by any of a number of methodsknown in the art. For example, in some embodiments, the polypeptide isisolated from the nutrient medium by conventional procedures including,but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, chromatography (e.g., ion exchange, affinity,hydrophobic interaction, chromatofocusing, and size exclusion), orprecipitation. In some embodiments, protein refolding steps are used, asdesired, in completing the configuration of the mature protein. Inaddition, in some embodiments, high performance liquid chromatography(HPLC) is employed in the final purification steps. For example, in someembodiments, methods known in the art, find use in the present invention(See e.g., Parry et al., Biochem. J., 353:117 [2001]; and Hong et al.,Appl. Microbiol. Biotechnol., 73:1331 [2007], both of which areincorporated herein by reference). Indeed, any suitable purificationmethods known in the art find use in the present invention.

Chromatographic techniques for isolation of the peroxidase polypeptideinclude, but are not limited to reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,are known to those skilled in the art.

In some embodiments, affinity techniques find use in isolating theimproved peroxidase oxidase enzymes. For affinity chromatographypurification, any antibody which specifically binds the peroxidasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with the peroxidase. The peroxidase polypeptidemay be attached to a suitable carrier, such as BSA, by means of a sidechain functional group or linkers attached to a side chain functionalgroup. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(Bacillus Calmette Guerin) and Corynebacterium parvum.

In some embodiments, the peroxidase variants are prepared and used inthe form of cells expressing the enzymes, as crude extracts, or asisolated or purified preparations. In some embodiments, the peroxidasevariants are prepared as lyophilisates, in powder form (e.g., acetonepowders), or prepared as enzyme solutions. In some embodiments, theperoxidase variants are in the form of substantially pure preparations.

In some embodiments, the peroxidase polypeptides are attached to anysuitable solid substrate. Solid substrates include but are not limitedto a solid phase, surface, and/or membrane. Solid supports include, butare not limited to organic polymers such as polystyrene, polyethylene,polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide,as well as co-polymers and grafts thereof. A solid support can also beinorganic, such as glass, silica, controlled pore glass (CPG), reversephase silica or metal, such as gold or platinum. The configuration ofthe substrate can be in the form of beads, spheres, particles, granules,a gel, a membrane or a surface. Surfaces can be planar, substantiallyplanar, or non-planar. Solid supports can be porous or non-porous, andcan have swelling or non-swelling characteristics. A solid support canbe configured in the form of a well, depression, or other container,vessel, feature, or location. A plurality of supports can be configuredon an array at various locations, addressable for robotic delivery ofreagents, or by detection methods and/or instruments.

In some embodiments, immunological methods are used to purify peroxidasevariants. In one approach, antibody raised against a variant peroxidasepolypeptide (e.g., against a polypeptide comprising any of SEQ ID NO: 4and/or 6, and/or an immunogenic fragment thereof) using conventionalmethods is immobilized on beads, mixed with cell culture media underconditions in which the variant peroxidase is bound, and precipitated.In a related approach, immunochromatography finds use.

In some embodiments, the variant peroxidases are expressed as a fusionprotein including a non-enzyme portion. In some embodiments, the variantperoxidase sequence is fused to a purification facilitating domain. Asused herein, the term “purification facilitating domain” refers to adomain that mediates purification of the polypeptide to which it isfused. Suitable purification domains include, but are not limited tometal chelating peptides, histidine-tryptophan modules that allowpurification on immobilized metals, a sequence which binds glutathione(e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitopederived from the influenza hemagglutinin protein; See e.g., Wilson etal., Cell 37:767 [1984]), maltose binding protein sequences, the FLAGepitope utilized in the FLAGS extension/affinity purification system(e.g., the system available from Immunex Corp), and the like. Oneexpression vector contemplated for use in the compositions and methodsdescribed herein provides for expression of a fusion protein comprisinga polypeptide of the invention fused to a polyhistidine region separatedby an enterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography;See e.g., Porath et al., Prot. Exp. Purif., 3:263-281 [1992]) while theenterokinase cleavage site provides a means for separating the variantperoxidase polypeptide from the fusion protein. pGEX vectors (Promega)may also be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toligand-agarose beads (e.g., glutathione-agarose in the case ofGST-fusions) followed by elution in the presence of free ligand.

Accordingly, in another aspect, the present invention provides methodsof producing the engineered enzyme polypeptides, where the methodscomprise culturing a host cell capable of expressing a polynucleotideencoding the engineered enzyme polypeptide under conditions suitable forexpression of the polypeptide. In some embodiments, the methods furthercomprise the steps of isolating and/or purifying the enzymepolypeptides, as described herein.

Appropriate culture media and growth conditions for host cells are wellknown in the art. It is contemplated that any suitable method forintroducing polynucleotides for expression of the enzyme polypeptidesinto cells will find use in the present invention. Suitable techniquesinclude, but are not limited to electroporation, biolistic particlebombardment, liposome mediated transfection, calcium chloridetransfection, and protoplast fusion.

Various features and embodiments of the present invention areillustrated in the following representative examples, which are intendedto be illustrative, and not limiting.

EXPERIMENTAL

The following Examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention. Indeed, there are various suitablesources for many of the reagents and equipment described below. It isnot intended that the present invention be limited to any particularsource for any reagent or equipment item.

In the experimental disclosure below, the following abbreviations apply:M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol(moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters);um and μm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) andhr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations perminute); psi and PSI (pounds per square inch); ° C. (degreesCentigrade); RT and rt (room temperature); RH (relative humidity); CV(coefficient of variability); CAM and cam (chloramphenicol); PMBS(polymyxin B sulfate); IPTG (isopropyl 13-D-1-thiogalactopyranoside); LB(Luria broth); TB (terrific broth); SFP (shake flask powder); CDS(coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid);nt (nucleotide; polynucleotide); aa (amino acid; polypeptide); E. coliW3110 (commonly used laboratory E. coli strain, available from the ColiGenetic Stock Center [CGSC], New Haven, Conn.); HTP (high throughput);HPLC (high pressure liquid chromatography); HPLC-UV (HPLC-UltravioletVisible Detector); 1H NMR (proton nuclear magnetic resonancespectroscopy); FIOPC (fold improvements over positive control); Sigmaand Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.; Difco (DifcoLaboratories, BD Diagnostic Systems, Detroit, Mich.); Microfluidics(Microfluidics, Westwood, Mass.); Life Technologies (Life Technologies,a part of Fisher Scientific, Waltham, Mass.); Amresco (Amresco, LLC,Solon, Ohio); Carbosynth (Carbosynth, Ltd., Berkshire, UK); Varian(Varian Medical Systems, Palo Alto, Calif.); Agilent (AgilentTechnologies, Inc., Santa Clara, Calif.); Infors (Infors USA Inc.,Annapolis Junction, Md.); and Thermotron (Thermotron, Inc., Holland,Mich.).

Example 1 E. coli Expression Hosts Containing Recombinant DyP Genes

The initial DyP enzymes used to produce the variants of the presentinvention were obtained from the wild-type peroxidase sequence Alr1585from cyanobacteria Anabaena (Nostoc) sp. (strain PCC 7120). SEQ ID NO: 2comprises the wild-type sequence. The wild-type DyP gene is a tetramerwith each monomer made up of ˜469 amino acids (53.3 kDa). TheDyP-encoding genes were cloned into the expression vector pCK110900(See, FIG. 3 of US Pat. Appln. Publn. No. 2006/0195947), operativelylinked to the lac promoter under control of the lad repressor. Theexpression vector also contains the P15a origin of replication and achloramphenicol resistance gene. The resulting plasmids were transformedinto E. coli W3110, using standard methods known in the art. Thetransformants were isolated by subjecting the cells to chloramphenicolselection, as known in the art (See e.g., U.S. Pat. No. 8,383,346 andWO2010/144103).

Example 2 Preparation of HTP DyP-Containing Wet Cell Pellets

E. coli cells containing recombinant DyP-encoding genes from monoclonalcolonies were inoculated into 180 μl LB containing 1% glucose and 30μg/mL chloramphenicol in the wells of 96-well shallow-well microtiterplates. The plates were sealed with 02-permeable seals and cultures weregrown overnight at 30° C., 200 rpm, and 85% humidity. Then, 10 μl ofeach of the cell cultures were transferred into the wells of 96-welldeep-well plates containing 390 mL TB and 30 μg/mL CAM. The deep-wellplates were sealed with 02-permeable seals and incubated at 30° C., 250rpm and 85% humidity until OD₆₀₀ 0.6-0.8 was reached. The cell cultureswere then induced by IPTG to a final concentration of 1 mM and incubatedovernight under the same conditions as originally used. The cells werethen pelleted using centrifugation at 4000 rpm for 10 min. Thesupernatants were discarded and the pellets frozen at −80° C. prior tolysis.

Example 3 Preparation of HTP DyP-Containing Cell Lysates

First, 400 μl lysis buffer containing 50 mM sodium phosphate buffer, pH6.5, 1 g/L lysozyme, and 0.5 g/L PMBS was added to the cell paste ineach well, produced as described in Example 2. The cells were lysed atroom temperature for 2 hours with shaking on a bench top shaker. Theplate was then centrifuged for 15 min at 4000 rpm and 4° C. The clearsupernatants were then used in biocatalytic reactions to determine theiractivity levels.

Example 4 Preparation of Lyophilized Lysates from Shake Flask (SF)Cultures

Selected HTP cultures grown as described above were plated onto LB agarplates with 1% glucose and 30 μg/ml CAM, and grown overnight at 37° C. Asingle colony from each culture was transferred to 6 ml of LB with 1%glucose and 30 μg/ml CAM. The cultures were grown for 18 h at 30° C.,250 rpm, and subcultured approximately 1:50 into 250 ml of TB containing30 μg/ml CAM, to a final OD₆₀₀ of 0.05. The cultures were grown forapproximately 195 minutes at 30° C., 250 rpm, to an OD₆₀₀ between0.6-0.8 and induced with 1 mM IPTG. The cultures were then grown for 20h at 30° C. and 250 rpm. The cultures were centrifuged 4000 rpm for 20min. The supernatant was discarded, and the pellets were resuspended in30 ml of 20 mM TRIS-HCl, pH 7.5. The cells were pelleted (4000 rpm for20 min) and frozen at −80° C. for 120 minutes. Frozen pellets wereresuspended in 30 ml of 20 mM TRIS-HCl pH 7.5, and lysed using aMicrofluidizer® processor system (Microfluidics) at 18,000 psi. Thelysates were pelleted (10,000 rpm×60 min), and the supernatants werefrozen and lyophilized to generate shake flake (SF) enzymes.

Example 5 Improvement in the Conversion of Amplex Red Substrate Comparedto SEQ ID NO: 4

SEQ ID NO: 4 was selected as the parent enzyme based on the results ofscreening variants for the conversion of the Amplex Red substrate. Ascompared to the wild-type sequence, SEQ ID NO: 4 comprises a residuedifference of D348N. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inExample 2, and the soluble lysate was generated as described in Example3. Each variant polypeptide was produced with a C-terminal his-tag.

For each variant, the cell pellet was lysed by adding 400 μL lysisbuffer containing 50 mM sodium phosphate buffer pH 6.5, 1 g/L lysozymeand 0.5 g/L PMBS and was shaken at room temperature for 2 hours on atable top shaker. The plates were centrifuged at 4000 rpm for 15 minutesat 4° C. to remove cell debris. In clear bottom UV-transparent flatbottom 96-well plates (UV-star plates), 200 μL HTP reactions were setupcomprising of 50 μL 32× diluted peroxidase HTP lysate, 150 μL of amastermix containing 66.7 mM Amplex Red (Sigma cat #: 90101), and 1.3 mMhydrogen peroxide (Sigma cat #: 216763) in 50 mM sodium phosphate bufferpH 6.5. For analysis, HTP reactions were analyzed by doing kinetic readsof the initial rate of the variants for 2 mins with 11 second intervalsper read at UV 560 nm using a Molecular Devices SpectraMax Plus 384spectrophotometer.

Activity relative to SEQ ID NO: 4 (Activity FIOP) was calculated as thepercent conversion of the product formed by the variant over the percentconversion produced by SEQ ID NO: 4. The results are shown in Table 5.1.

TABLE 5.1 Activity of Variants Relative to SEQ ID NO: 4 FIOP¹ (Activity)SEQ ID Amino Acid Differences (Relative to SEQ NO: (nt/aa) (Relative toSEQ ID NO: 4) ID NO: 4)  5/6 N393D +++  7/8 Q208N +++  9/10 V206L +++ 11/12 Y307D +++  13/14 I160V; Y307C +++  15/16 P311V +++  17/18 T306A+++  19/20 C224P +++  21/22 Y307R +++  23/24 R25A; N29A; H43Y +++  25/26E128K; Y307N +++  27/28 T171V; L260R ++  29/30 Y307S ++  31/32 R25Q;N29A; S65G; F82K; D92G ++  33/34 N191D ++  35/36 Y307M ++  37/38 V221Y++  39/40 Y307L ++  41/42 N191S; L260R ++  43/44 Y307C ++  45/46 V221T++  47/48 L301S ++  49/50 F202W ++  51/52 T306V ++  53/54 L301V ++ 55/56 N168W ++  57/58 D185Q ++  59/60 I160H; I188V ++  61/62 E127K;L301I ++  63/64 T332A ++  65/66 V221L ++  67/68 N191S; A469V ++  69/70E128K; Y307Q ++  71/72 C224R ++  73/74 C224L ++  75/76 E80K; E128K;T306F ++  77/78 Y307E ++  79/80 V221H ++  81/82 K228M; L260R ++  83/84D185E; Y307H ++  85/86 N191S ++  87/88 S90Y ++  89/90 D185T ++  91/92V221S ++  93/94 N191G +  95/96 A367G +  97/98 C224Y +  99/100 Y307G +101/102 V221P + 103/104 I160M + 105/106 V221M + 107/108 C224R; L260R +109/110 E241K; D270N; N283T; T306L + 111/112 K228G + 113/114 N222E +115/116 L143R + 117/118 N222S + 119/120 L164D + 121/122 L143H + 123/124L260R; Y307T + 125/126 F226V + 127/128 N168S + 129/130 L301W + 131/132I160R + 133/134 V221R + 135/136 N26E; N29K + 137/138 E127K; N168S +139/140 V290L + 141/142 N191A + 143/144 N168H + 145/146 N168K + 147/148P311I + 149/150 T306K + 151/152 N222V + 153/154 V221D + 155/156 S90Q +157/158 L143S + 159/160 L164K + 161/162 N168M + 163/164 N222G + 165/166F226I + 167/168 L143V + 169/170 L189W; R190V; F226P + ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 4 and defined as follows: “+” 1.50 to 2.25, “++” 2.25 to3.00, “+++” >3.00

Example 6 Improvement in the Conversion of Amplex Red Substrate Comparedto SEQ ID NO: 6

SEQ ID NO: 6 was selected as the next parent enzyme, based on theresults described in Example 5. Libraries of engineered genes wereproduced using well-established techniques (e.g., saturation mutagenesisand recombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inExample 2, and the soluble lysate was generated as described in Example3.

For each variant, the cell pellet was lysed by adding 400 μL lysisbuffer containing 50 mM sodium phosphate buffer pH 6.5, 1 g/L lysozymeand 0.5 g/L PMBS and was shaken at room temperature for 2 hours on atable top shaker. The plates were centrifuged at 4000 rpm for 15 minutesat 4° C. to remove cell debris. In clear bottom UV-transparent flatbottom 96-well plates (UV-star plates), 200 μL HTP reactions were setupcomprising of 50 μL 32× diluted peroxidase HTP lysate, 150 μL of amastermix containing 66.7 mM Amplex Red (Sigma cat #: 90101), and 1.3 mMhydrogen peroxide (Sigma cat #: 216763) in 50 mM sodium phosphate bufferpH 6.5. For analysis, HTP reactions were analyzed by doing kinetic readsof the initial rate of the variants for 2 mins with 11 second intervalsper read at UV 560 nm using a Molecular Devices SpectraMax Plus 384spectrophotometer.

The percent conversion relative to SEQ ID NO: 6 (Percent ConversionFIOP) was calculated as the percent conversion of the product formed bythe variant over the percent conversion produced by SEQ ID NO: 6. Theseresults are shown in Table 6.1. The percent conversion was calculated bydividing the area of the product peak by the sum of the areas of thesubstrate, product and impurities/side product peaks as observed by theHPLC analysis.

TABLE 6.1 Activity of Variants Relative to SEQ ID NO: 6 FIOP¹ (Activity)SEQ ID Amino Acid Differences (Relative to SEQ NO: (nt/aa) (Relative toSEQ ID NO: 6) ID NO: 6) 171/172 N26A; Y307M +++ 173/174 N26E; V221Y +++175/176 Q208N; T306V +++ 177/178 S90Y +++ 179/180 N26E; N191S +++181/182 N26A; V221H +++ 183/184 N26A; L301S; Y307G ++ 185/186 N191S ++187/188 V221S ++ 189/190 S90Y; C224P ++ 191/192 V221Y ++ 193/194 Y307M++ 195/196 F202W; V221S ++ 197/198 N26E; L301S ++ 199/200 Y104H; V221H++ 201/202 N26A; Y307Q ++ 203/204 N26E; N191G ++ 205/206 N26E; V221H;Y307S ++ 207/208 N26A; V221Y ++ 209/210 Y307L ++ 211/212 Y307Q ++213/214 N26A; N191S ++ 215/216 N26A; L301S ++ 217/218 N168S; T306A ++219/220 F226V ++ 221/222 N191S; Y307E ++ 223/224 F202W ++ 225/226 S90Y;T306S + 227/228 N26E; V221L + 229/230 E80K; Q208N; T306F + 231/232 N26A;N191S F202W; V221T; Y307G + 233/234 N26E; N191S; Y307M + 235/236 N26E;E80K; L301S + 237/238 F202L + 239/240 V221T; Y307E + 241/242 N191D;V221M; Y307G + 243/244 N26A; F202W; V221Y + 245/246 S90Y; T306V +247/248 N26E; N191S; Y307E + 249/250 F202W; V221H + 251/252 N26E;V221M + 253/254 N26A; N191G; V221Y + 255/256 E128K; F202W; V221S +257/258 E80K; N191S + 259/260 F202W; V221T + 261/262 L301S + 263/264S90Y; T306W + 265/266 Y307D + 267/268 N168S; Q208N + 269/270 L301S;Y307K + 271/272 N26E; N191G; V221S + 273/274 N26A; N191G + 275/276 E80K;A81T; F226V + 277/278 N168S; Q208N; C224L; T306V + 279/280 N191G +281/282 V221L; L301S + 283/284 N26A; V221S + 285/286 Y104D; N168W +287/288 E80K; F202W; V221L; L301S; Y307G + 289/290 N26E; 1160V; F267L;Y307M; H329Y + ¹Levels of increased activity were determined relative tothe reference polypeptide of SEQ ID NO: 6 and defined as follows: “+”1.90 to 2.50, “++” 2.50 to 3.50, “+++” >3.50

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1. An engineered peroxidase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4 and/or 6, or a functional fragment thereof, wherein said engineered peroxidase comprises at least one substitution or substitution set in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4 and/or
 6. 2. The engineered peroxidase of claim 1, wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4, and wherein said engineered peroxidase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 393, 25/29/43, 25/29/65/82/92, 26/29, 80/128/306, 90, 127/168, 127/301, 128/307, 143, 160, 160/188, 160/307, 164, 168, 171/260, 185, 185/307, 189/190/226, 191, 191/260, 191/469, 202, 206, 208, 221, 222, 224, 224/260, 226, 228, 228/260, 241/270/283/306, 260/307, 290, 301, 306, 307, 311, 332, and 367, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:
 4. 3. The engineered peroxidase of claim 1, wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 6, and wherein said engineered peroxidase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 393, 25/29/43, 25/29/65/82/92, 80/128/306, 90, 127/301, 128/307, 143, 160, 160/188, 160/307, 164, 168, 171/260, 185, 185/307, 191, 191/260, 191/469, 202, 206, 208, 221, 222, 224, 224/260, 228, 228/260, 241/270/283/306, 260/307, 301, 306, 307, 311, 332, and 367, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:
 6. 4. The engineered peroxidase of claim 1, wherein said engineered peroxidase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered peroxidase variant set forth in Table 5.1 and/or 6.1
 5. The engineered peroxidase of claim 1, wherein said engineered peroxidase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered peroxidase variant set forth in SEQ ID NOS: 4 and/or
 6. 6. The engineered peroxidase of claim 1, wherein said engineered peroxidase is a variant engineered peroxidase set forth in SEQ ID NOS: 4 and/or
 6. 7. The engineered peroxidase of claim 1, wherein said engineered peroxidase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered peroxidase variant set forth in the even numbered sequences of SEQ ID NOS: 6-290.
 8. The engineered peroxidase of claim 1, wherein said engineered peroxidase comprises a polypeptide sequence set forth in the even numbered sequences of SEQ ID NOS: 6-290.
 9. The engineered peroxidase of claim 1, wherein said engineered peroxidase comprises at least one improved property compared to wild-type Anabaena (Nostoc) sp. peroxidase.
 10. The engineered peroxidase of claim 9, wherein said improved property comprises improved activity on a substrate.
 11. The engineered peroxidase of claim 10, wherein said substrate comprises 10-acetyl-3,7-dihydroxyphenoxazine.
 12. The engineered peroxidase of claim 1, wherein said engineered peroxidase is purified.
 13. A composition comprising at least one engineered peroxidase of claim
 1. 14. A polynucleotide sequence encoding at least one engineered peroxidase of claim
 1. 15. A polynucleotide sequence encoding at least one engineered peroxidase, wherein said polynucleotide sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOS: 3 and/or 5, wherein the polynucleotide sequence of said engineered peroxidase comprises at least one substitution at one or more positions.
 16. A polynucleotide sequence encoding at least one engineered peroxidase comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOS: 3 and/or 5, or a functional fragment thereof.
 17. The polynucleotide sequence of claim 15, wherein said polynucleotide sequence is operably linked to a control sequence.
 18. The polynucleotide sequence of claim 15, wherein said polynucleotide sequence is codon optimized.
 19. The polynucleotide sequence of claim 15, wherein said polynucleotide comprises an odd-numbered sequence of SEQ ID NOS: 5-289.
 20. An expression vector comprising at least one polynucleotide sequence of claim
 15. 21. A host cell comprising at least one expression vector of claim
 20. 22. A host cell comprising at least one polynucleotide sequence of claim
 15. 23. A method of producing an engineered peroxidase in a host cell, comprising culturing the host cell of claim 21, under suitable conditions, such that at least one engineered peroxidase is produced.
 24. The method of claim 23, further comprising recovering at least one engineered peroxidase from the culture and/or host cell.
 25. The method of claim 24, further comprising the step of purifying said at least one engineered peroxidase. 